Expression screening vector

ABSTRACT

An expression vector for preparing a library of an antibody variable region, which can express comprising polypeptides containing the H-chain and/or L-chain variable regions of antibodies in the membrane-bound form on the surfaces of eukaryotic cells, which is containing the nucleotide sequence of AKL (formula 1) and/or the nucleotide sequence AKH (formula 2), and which is replicable in the cells: 
     
       
         5′-P L -S L -C L -M L -A L -3′  (formula 1)(AKL) 
       
     
     
       
         5′-P H -S H -C H -M H -A H -3′  (formula 2)(AKH) 
       
     
     wherein P L  and P H  represent a promoter, respectively; S L  and S H  represent a nucleotide sequence coding for a signal peptide respectively; C L  represents a nucleotide sequence coding for the L-chain constant region of an antibody; C H  represents a nucleotide sequence coding for the H-chain constant region of an antibody or a nucleotide sequence coding for a polypeptide containing at least CH1 of the H-chain constant region of an antibody; A L  and A H  represent a polyadenylation signal, respectively; “5′-” and “-3′” refer to the 5′ side and the 3′ side, respectively, of the nucleotide sequence; and M L  and M H  represent a nucleotide sequence coding for the transmembrane domain, respectively, while either M L  or M H  may be a mere chemical bond when the expression vector contains the nucleotide sequence of AKL and AKH. Cloning site of R1 L , R2 L , R1 H  and R2 H  is present within or in the vicinity of S L , C L , S H  and C H  respectively, in order for the nucleotide sequences coding for the L-chain and H-chain variable regions to be readily inserted in between S L  and C L , and S H  and C H  respectively.

This application is a 317 of PCT/JP94/02033, filed on Dec. 2, 1994.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a vector which directs expression of anantibody or a portion of antibody at least containing an antigen bindingsite (hereinafter designated as antibodies) on the surface of cellmembrane, in order to select a nucleotide sequence coding for a variableregion of the antibody for a specific antigen (hereinafter designated asnucleotide sequence of variable region), a library and a method forselecting a nucleotide sequence of antigen-specific antibody variableregion using the said vector, and a kit containing the same.

2. Description of the Related Art

Development of antibody for application in pharmaceuticals has recentlyprogressed. Antibody of those pharmaceutical applications is preferablya human type due to antigenicity to the humans. However, preparation ofhuman antibody to the specific antigen has not been established unlikemouse monoclonal antibody with established hybridoma method. From thesepoints of view, a development for a new process applicable to thepreparation of human monoclonal antibody in place of hybridoma method,has been expected.

Preparation of specific antibody for specific antigen by means ofgenetic engineering process, requires a nucleotide sequence of variableregions of the H- and L-chains which determine the antigen specificityof the antibody. Genetically engineered preparation of antibody usingthe variable region of the H- and L-chains has been known [Xiang, J. etal. (1990), Mol. Immun., 27, 809; Bebbington, C. R. et al. (1992),Biotechnology, 10, 169].

A method for preparing the nucleotide sequence of variable region ofantigen-specific H- and L-chains, using E. coli phage or phagemid hasbeen known [Huse. W. D. et al. (1989). Science, 246, 1275; McCafferty,J. et al. (1990), Nature, 348, 552 and Kang, A. S. et al. (1991), Proc.Natl. Acad. Sci. USA 88, 44363].

In these methods, antibody library is prepared by producing Fab (referto FIG. 1) per se, or antibody library (phage antibody library) isprepared by producing fused phage coat protein with Fab or a singlechain Fv, which is prepared by linking the H-chain variable region tothe L-chain variable region of antibody with suitable linker (refer toFIG. 1, hereinafter designates as scFv), thus antigen-specific antibodyand gene thereof are selected by means of binding affinity with antigen.

As explained hereinabove, phage antibody library is prepared byproducing Fab or scFv by E. coli. Fab or scFv produced by E. coli forman inclusion body (in case of intracellular production), or isaccumulated in the periplasm to form insoluble protein (in case ofsecretion) [Anand, N. N. et al. (1991), J. Biol. Chem., 266, 21874;Huston, J. S. et al. (1991), Methods Enzymol., 203, 46]. Inclusion bodyand insoluble protein should be solubilized to construct higher-orderstructure (renature) for obtaining protein with antigen-bindingactivity.

Preparation of scFv by linking the H-chain variable region to L-chainvariable region with suitable linker in various antibodies, for whichproperties thereof were well known, using expression system of E. colihas been tried [Bird, R. E. et al. (1991), TIBTEC, 9, 132; Anand, N. N.et al. (1991), J. Biol. Chem., 266, 21874]. Affinity of the scFv isgenerally decreased as compared with that of Fab obtained by papainhydrolysis. Further productivity of scFv varies greatly by an order ofthe H- and L-chain variable regions [Tsumoto, K. et al. (1994), Biochem.Biophys. Res. Comm., 201, 546]. Therefore production of scFv of someantibodies by E. coli may show disadvantages. In the phage antibodysystem hereinbefore explained, the scFv is prepared to form fusedprotein with minor coat protein g3p of fd phage. Fused protein of somekind of scFv loses antigen-binding activity [Furuta, S. et al., apresentation at Japan. Biochem. Soc. Meeting, 1994].

As explained hereinabove, the expression of scFv or phage antibody usingscFv in E. coli has various disadvantages and the case with mere successin preparation of an antibody does not warrant the success in all othercases of the antibodies. There are possibilities to be preparedincomplete library having problems such that the expressed variableregion has a bias in variety and that affinity of the expressed antibodydiffers from that of the original.

A problem in expression of scFv in E. coli seems to be caused byartificial structure linking the H-chain variable region to the L-chainvariable region with linker and, in addition to that, is thought to bemainly caused by cellular structure of E. coli which has no organellafor constructing higher-order structure of protein such as endoplasmicreticulum in animal cells. Originally, since the active antigen-bindingsite of antibody can be formed by establishing exact steric structurecontaining intramolecular disulfide bonds in H-chain and L-chain,respectively, and by forming the one to one corresponding dusulfide bondof the H- and L-chains,the higher-order structure of protein isextremely important for the expression of activity.

Expression system for Fab on the surface of phage in place of scFv hasthe same problem. Considering the large size of molecular weight of Fabof about 45,000 as compared with that of scFv of about 26,000, andnecessity of exact association with two polypeptide chains, anexpression of Fab having exact activity will be more difficult than thatof scFv. Expression of active Fab in E. coli shows less productivity[Skerra, A. et al. (1991) Protein Eng. 4, 971].

As explained in the above, a screening system in use of E. coli is notpreferable for expression of protein having complex higher-orderstructure such as antibody which is originally produced in animal cells.Accordingly, the expression screening system in use of eukaryotic cells,especially animal cells, is thought to be preferable. Expression systemin use of animal host cells shows no problems as like in the expressionsystem in E. coli [Wood, C. R. et al., (1990) J. Immunol., 145, 3011]. Alibrary which expresses exact antigen-binding activity of the originalantibody could be prepared, and the nucleotide sequence of variableregion of antigen specific antibody could be screened effectively fromthe library. Natural human antibody is preferable for antibodytherapeutics in view of antigenicity in human. A process for productionof human antibody by means of recombinant DNA technology in use ofanimal host cells is preferable.

On the point that the final expression is performed by animal cells, useof animal cells at the screening step may be preferable, in order not tomake difference in antigen-binding activity of antibody in the screeningsteps and the production steps.

As clearly explained hereinabove, although the screening system in E.coli for nucleotide sequence of variable region of antigen-specific H-and L-chain is known, development on more preferable screening system inuse of eukaryotic cells, specifically animal cells is expected.

SUMMARY OF THE INVENTION

The present invention relates to a method for screening nucleotidesequence of variable region of antigen-specific antibody in use ofeukaryotic cells, specifically animal cells, which are preferable forexpression of antibody, and a vector therefor.

The inventors of the present invention have tried to find out the ideacomprising preparing a vector which expresses antibodies on cellmembrane, inserting nucleotide sequence of variable region of variousantibody to the vector to express the antibodies on the cell membrane,concentrating the cells which express the antibodies binding to antigenin use of marker of antigen-binding activity, and obtaining thenucleotide sequence of variable region of antigen-specific antibody.

The present invention consists of the following concepts.

(1) An expression vector for preparing a library of an antibody variableregion, which can express polypeptides containing the H-chain and/orL-chain variable regions of antibodies in the membrane-bound form on thesurfaces of eukaryotic cells, and which is containing the nucleotidesequence of AKL (formula 1) and AKH (formula 2) and replicable in thecells:

5′-P_(L)-S_(L)-C_(L)-M_(L)-A_(L)-3′  (formula 1)(AKL)

5′-P_(H)-S_(H)-C_(H)-M_(H)-A_(H)-3′  (formula 2)(AKH)

wherein P_(L) and P_(H) represent a promoter, respectively; S_(L) andS_(H) represent a nucleotide sequence coding for a signal peptide,respectively; C_(L) represents a nucleotide sequence coding for theL-chain constant region of an antibody; C_(H) represents a nucleotidesequence coding for the H-chain constant region of an antibody or anucleotide sequence coding for a polypeptide containing at least CH1 ofthe H-chain constant region of an antibody; A_(L) and A_(H) represent apolyadenylation signal, respectively; “5′-” and “-3′” refer to the 5′side and the 3′ side, respectively, of the nucleotide sequence; andM_(L) and M_(H) represent a nucleotide sequence coding for thetransmembrane domain, respectively while either M_(L) or M_(H) may be amere chemical bond when the expression vector contains the nucleotidesequence of AKL and AKH. Cloning site of R1_(L), R2_(L), R1_(H) andR2_(H) is present within or in the vicinity of S_(L), C_(L), S_(H) andC_(H), respectively, in order for the nucleotide sequences coding forthe L-chain and H-chain variable regions to be readily inserted inbetween S_(L) and C_(L), and S_(H) and C_(H), respectively.

(2) The expression vector according to the above (1) wherein the saidcloning site of R1_(L), R2_(L), R1_(H) and R2_(H) is selected fromrecognition sequence of restriction enzymes [MunI, AclI, BspLU11I, MluI,BssHII, NheI, XbaI, SplI, Bspl407I, ClaI, XhoI, SalI and Afl II].

(3) The expression vector according to (1) wherein the said nucleotidesequence coding for the transmembrane domain of M_(L) and M_(H) is anucleotide sequence coding for the transmembrane domain ofthrombomodulin.

(4) The expression vector according to (3) wherein the said expressionvector contains the nucleotide sequence of AKL and AKH, and the cloningsite of R1_(L), R2_(L), R₁ _(H) and R2_(H) is the recognition sequenceof XhoI, SplI, ClaI and MluI, respectively, and M_(L) is a mere-chemicalbond.

(5) The expression vector according to (3) wherein the said expressionvector contains the nucleotide sequence of AKL and AKH, and the cloningsite of R1_(L), R2_(L), R1_(H) and R2_(H) is the recognition sequence ofXhoI, SpeI, BamHI and EcoRI, respectively, and M_(L) is a mere chemicalbond.

(6) The expression vector according to (3) wherein the said expressionvector contains the nucleotide sequence of AKL and AKH, and the cloningsite of R1_(L), R2_(L), R1_(H) and R2_(H) is the recognition sequence ofXhoI, SpeI, BamHI and ApaI, respectively, and M_(L) is a mere chemicalbond.

(7) The expression vector according to (1) wherein the said vector isreplicable in COS cells.

(8) A vector, which is used for preparing a library of antibody variableregion, being inserted a large number of nucleotide sequences coding forH-chain variable regions of antibodies and/or nucleotide sequencescoding for L-chain variable regions of antibodies into the cloning sitesof the expression vector of (1).

(9) A group of eukaryotic cells comprising being expressed polypeptidescontaining the H-chain and L-chain variable regions of antibodies in themembrane-bound form on the surface of the cells by introducing thevectors containing the vector of (8) to the host cells.

(10) A method for selecting nucleotide sequences coding for antibodyvariable regions binding to a specific antigen from the nucleotidesequences coding for a large number of antibody variable regions,comprising:

(a) isolating the cells bound to said antigen from the group ofeukaryotic cells of (9), and

(b) recovering the expression vector from the isolated cells to obtainnucleotide sequences coding for antibody variable regions bound to theantigen.

(11) The method according to (10) comprising immobilizing the antigen onthe surface of the solid, and isolating the cells by adhering the cells,which express the antigen-binding polypeptide, to the immobilizedantigen.

(12) The method according to (10) comprising labelling the antigen withfluorescent substance, biotin or magnetic beads and isolating the cellswhich express the antigen-binding polypeptide by flow cytometry orimmunomagnetic beads method.

(13) A screening kit for nucleotide sequences coding for variableregions of the antigen-specific antibody comprising the expressionvector of (1) or (8), host cells and auxiliary components.

As illustrated in a schematic representation of IgG1 in FIG. 1, antibodyconsists of two large and small polypeptides which are designated as“H-chain” for the large chain and “L-chain” for the small one. Eachchain consists of a “variable region” for antigen-binding site inN-terminal region, and the fixed “constant region” depending upon theantibody classes. The constant region in H-chain consists of fourdomains and a domain in the N-terminal end is designated as “CH1”.

“Promoter P_(L) and P_(H)” and “nucleotide sequences S_(L) and S_(H)coding for signal peptide” in FIG. 1 and FIG. 2 are workable ineukaryote.

“The transmembrane domain” in the present invention means thetransmembrane domain consisting of 15-30 amino acids abundant inhydrophobic amino acids and a region having anchor region consisting ofcharged amino acids in carboxyl terminus for positioning the carboxylterminus to the cytoplasmic region. The “membrane-bound form” in thepresent invention means that antibodies have a transmembrane domain inthe part of the antibodies.

A term “replicable in the cells” in the present invention means that thevector can be autoreplicable independently from the host chromosome whenit is transduced into prokaryotic cells such as E. coli or eukaryoticcells such as animal cells. To be more concrete, the autoreplication inE. coli can be achieved by recombinating the ori from pBR322 or pUC18 inthe vector, or autoreplication in COS cells can be performed byrecombinating the ori from SV40 in the vector.

A term “mere chemical bond” means phosphodiester bond binding 5′-carbonin deoxyribose of a nucleotide to 3′-carbon in deoxyribose of the nextnucleotide. “COS cells” includes COS1 cell (ATCC CRL 1650) and COS7cell(ATCC CRL 1651). A term “auxiliary components” includes E. coli forhost cell, and reagents and buffer solution used for reactions such asrestriction enzyme digestion, DNA ligation, gene transduction andantigen labelling. Antibody is a tetrameric protein composed of twoheavy (H) chains and two light (L) chains of peptides (refer to FIG. 1).Each chain consists of a variable region which makes up theantigen-binding site and a constant region which determines theimmunoglobulin class to which the molecule belongs. Antigen specificityof antibody is determined by combination of H-chain and L-chain variableregions. Genetic engineering process for preparation of antigen specificantibody requires at least H-chain and L-chain variable regionnucleotide sequence of antigen specific antibody.

Consequently, an object of the present invention is to provide ascreening method for nucleotide sequence of variable region ofantigen-specific antibody in use of expression system of eukaryoticcells, specifically animal cells.

We have found that a method for obtaining nucleotide sequence ofvariable region of antigen-specific antibody comprising expressingantibodies on the host cell membrane, and selecting and isolating thehost cells which express antigen-specific antibodies by means ofantigen-binding activity of the antibodies which are expressed on themembrane.

Antibodies consist of secretory type and membrane-bound type, the latterof which is expressed on the cell membrane as a B cell receptor andwhich is known to require the other subunit depending on its class forexpression on the membrane [Venkitaraman, A. K. et al., (1991), Nature,352, 777]. We have had an idea that the constant region of theantibodies was made in secretory type for possible expression of theantibodies per se, and the transmembrane domain of the membrane proteinother than antibody was linked to carboxyl terminus of H-chain and/orL-chain of the antibodies for expressing the antibodies on the cellmembrane.

In case that any one of a nucleotide sequence of AKL of the formula 1and AKH of the formula 2 is integrated in the vector, the chain, whichis not integrated therein (for example, H-chain in case of AKL in thevector, and L-chain in case of AKH in the vector), is previouslyincorporated in the host cells, or should be integrated in the hostcells by cotransduction of the other vector containing the chain.

Preparation of plasmid vector pSEL, an example of a vector whichexpresses antibodies on the cell membrane, is illustrated in Example 1.Plasmid vector pSEL has been deposited in National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology, Ministry of International Trade and Industry, as depositionE. coli: JM109-pSEL (FERM BP-4896) on Nov. 18, 1994.

In the pSEL, human IgG1 constant region nucleotide sequence (Cγ1) for anucleotide sequence coding for H-chain constant region (hereinafterdesignates as constant region nucleotide sequence), human κ chainconstant region nucleotide sequence (Cκ) for L-chain constant regionnucleotide sequence, and transmembrane domain nucleotide sequence ofhuman thrombomodulin (TM) are inserted and TM is ligated at 3′ terminusof Cγ1. Cloning sites are constructed for 5′ terminus of H-chainvariable region nucleotide sequence by introducing BamHI site in oneside, and for 3′ termions of the H-chain variable region nucleotidesequence in use of the originally located ApaI site at the neighbouringwith 5′ terminus of Cγ₁ in the other side. In the L-chain, XhoI site isnewly introduced at 5′ terminus and, for 3′ terminus, SpeI site is newlyintroduced in the neighbouring with 5′ terminus of Cκ. Genes of H-chainand L-chain are located at down stream of each promoter and arranged intandem for the same direction. The pSEL contains ori from pBR322 forautoreplication in E. coli, and ori from SV40 for autoreplication in COScells.

Examples of vectors which express the antibodies on cell membrane areillustrated in Examples 2 and 3 for preparation of plasmid vector pSEand plasmid vector pSE 2. The plasmid vector pSE has been deposited inNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology, Ministry of International Trade andIndustry as deposition E. coli: JM109-pSE (FERM BP-4894), and theplasmid vector pSE2 has been deposited in the same institution asdeposition E. coli: JM109-pSE2 (FERM BP-4895) on Nov. 18, 1994.

The plasmid vector pSE is basically the equivalent vector to pSEL exceptthe original vector is pUC18, and ApaI cloning site in pSEL is replacedby EcoRI cloning site. In pSE2, cloning sites are replaced as follows:

BamHI→ClaI, EcoRI→MluI and SpeI→SplI.

Primary condition on a restriction enzyme at cloning site for cloningnucleotide sequence of human antibody variable region is that therecognition sites of the restriction enzyme should not be located atleast in human germ line V_(H), D, J_(H) and J_(L) genes. Hithertoreported human germ line V_(H), D, J_(H) and J_(L) genes were searchedin the references [SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST FIFTHEDITION (1991), U.S. Department of Health and Human Services, PublicHealth Service National Institutes of Health; Schable, K. F. et al.(1993) Biol. Chem. Hoppe-Seyler, 374, 1001, Williams, S. C. et al.(1993) Eur. J. Immunol., 23, 1456; Matsuda, F. et al.(1993) NatureGenetics, 3, 88; Cook, G. P. et al. (1993) Nature Genetics, 7, 162] andthe data base (GenBank and EMBL), and restriction sites were retrieved.The recognition sites of restriction enzymes such as MunI, AclI,BspLU11I, MluI, BssHII, NheI, XbaI, SplI, Bspl407I, ClaI, XhoI, SalI andAfl II were not found to exist in the known human germ line V_(H), D,J_(H), V_(L) and J_(L) genes. Consequently, these recognition sites ofthe enzyme can be used as cloning sites. Cloning site for pSE2 isselected from the above commercially available enzymes.

Separation of the nucleotide sequence of variable region is performed byPCR (polymerase chain reaction). Amplification and separation ofnucleotide sequences of variable region of antibodies by PCR have beenknown [Orlandi, R. et al. (1989), Proc. Natl. Acad. Sci. USA, 86, 3833].Preparation of nucleotide sequence of variable region of mousemonoclonal antibody M21 for rat IgG2b and expression thereof in COS7cells are illustrated in Example 4.

Examples of primers for amplification of nucleotide sequences of mouseH-chain variable region are shown in Table 1. Examples of primers foramplification of nucleotide sequences of mouse κ chain variable regionare shown in Table 2.

Variable region of antibody is composed of framework region andhypervariable region, and is classified into subgroups according tohomology of amino acid sequence in the framework region [SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST FIFTH EDITION (1991), U.S. Departmentof Health and Human Services, Public Health Service National Institutesof Health].

Conserved sequence located at 5′ terminus of the variable region in thesubgroup is used as a reverse primer. Complementary strand nucleotidesequence from 3′ terminus of mouse J_(H) and J_(K) genes to 5′ terminusof Cγ₁ and Cκ is used as a forward primer. The reverse primer sequenceand the forward primer sequence in H-chain variable region have BamHIsite and ApaI site, respectively. The reverse primer sequence and theforward primer sequence in L-chain variable region have XhoI site andSpeI site, respectively. PCR product of H-chain variable region and pSELare digested by BamHI and ApaI and ligated to insert nucleotide sequenceof the H-chain variable region into pSEL. Similarly, PCR product ofL-chain variable region and pSEL are digested by XhoI and SpeI, andligated to insert nucleotide sequence of the L-chain variable regioninto pSEL.

Preparation of a library of antibody variable region is exemplified inExample 5. Nucleotide sequences of the variable region are amplified andseparated by PCR from various antibody-producing cells, and the libraryof the antibody variable region is prepared by inserting them in pSEL asshown in Example 4. Since nucleotide sequences of H-chain and L-chainvariable region are isolated independently thereafter combined on theplasmid, antibody repertoire which has not been existed in vivo ispossibly incorporated in the library.

Selection of nucleotide sequences of antibody variable region having theantigen sepcificiy is illustrated in Example 6. Vector DNA of thelibrary to be selected is introduced into host cells by electroporation,DEAE dextran method and others. In Example 6, the electroporation isapplied. Electroporation is described in the experimental books (SeriesBiochemical Experiments 1, Methods in Research Studies on Genes III,Chapter 15, Electroporation, Igarashi, T. et al.). On two to three daysculture aftertransduction of the vector DNA, the antibodies areexpressed on the host cell membrane. The host cells which expressantigen-binding antibodies on the membrane are separated by means ofindicator of antigen-binding activity. Examples of separation methodsare a panning method in which antibody expression cells are plated onthe surface of antigen-bound plastic plate and the cells expressedantigen-binding antibody on the surface are adhered and separated, and acell sorting method by flow cytometry in which antigen-binding antibodyexpressed cells are specifically stained by an antigen previouslylabelled with fluorescent FITC or biotin. The panning method isdescribed in “Biomanual Series 3, Gene Cloning” [Yokota, T. and Arai, K.Ed. (1994). Yodosha Publ.]. The sorting method is described in “Flowcytometry-Technique and Practice” [Ed. Ota, K. and Nomura, K. (1984),Kani Publ. Co.]. Vector DNA is recovered from separated host cells bymeans of Hirt method [Hirt, B. (1967), J. Mol. Biol., 26, 365]. Aprocedure of the above concentration operation is repeated for severalprocesses to obtain nucleotide sequence of antigen-specific antibodyvariable region. In Example 6, one operation results in 54-foldconcentration, however superior effective concentration could beachieved by the improvement of the conditions, such as sortingconditions.

Hereinabove exemplified processes are illustrations of a model system inuse of mouse monoclonal antibody M21 and its antigen rat IgG2b anti L3T4antibody GK1.5. Examples 7 and 8 hereinbelow are exemplified by showinga preparation of a library of human antibody variable region in use ofpSE plasmid, and experimental data of screening of human anti-HBsantibody.

In Example 7, peripheral blood lymphocytes of volunteer who is anti-HBsantibody positive are used as a gene source. Human antibody variableregion nucleotide sequences are isolated by PCR and inserted in aplasmid pSE to prepare a library of human antibody variable regioncomposed of approximately 5×10⁶ colonies. Examples of primer fornucleotide suquences of human H-chain variable region are shown in Table4 and those for nucleotide sequences of human κ chain variable regionare shown in Table 5.

Variable region of antibody is composed of framework region andhypervariable region, and is classified into subgroups according tohomology of amino acid sequence in the framework region [SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST FIFTH EDITION (1991). U.S. Departmentof Health and Human Services, Public Health Service National Institutesof Health].

Conserved sequence located at 5′ terminus of the variable region in thesubgroup is used as a H-chain reverse primer. Complementary strandnucleotide sequence of 3′ terminus of human J_(H) gene is used as aH-chain forward primer. A complementary strand nucleotide sequence from3′ terminus of J_(K) gene to 5′ terminus of Cκ gene is used as a L-chainforward primer. The reverse primer sequence and the forward primersequence in H-chain variable region have BamHI site and EcoRI site,respectively. The reverse primer sequence and the forward primersequence in L-chain variable region have XhoI site and SpeI site,respectively. PCR product of H-chain variable region and pSE aredigested by BamHI and EcoRI, and ligated to insert nucleotide sequenceof the H-chain variable region into pSE. Similarly, PCR product ofL-chain variable region and pSE are digested by XhoI and SpeI, andligated to insert nucleotide sequence of the L-chain variable regioninto pSE.

In Example 8, clones bound with recombinant HBs antigen are isolated bystarting from approximately 3×10⁷ COS7 cells from the library, andconcentrating three times with biotin-labelled recombinant HBs antigen.Nucleotide sequences of H-chain variable region and κ chain variableregion are obtained from the thus obtained clones, and are, for example,introduced into COS cells after inserting those sequences into secretoryantibody-producing vector in which TM site is removed from pSE plasmidin FIG. 4 to prepare human anti-HBs monoclonal antibody by well knownmethod [Xiang, J. et al. (1990), Mol. Immun., 27, 809; Bebbington, C. R.et al. (1992), Bio/technology, 10, 169].

As explained hereinabove, nucleotide sequences of variable region inantigen-specific antibody can be screened by a method of the presentinvention in case of animals such as mouse and human wherein detailedinformations on nucleotide sequence of antibody variable region areavailable. These screenings can easily be performed by use of thescreening kit consisting of the expression vectors, host cells andauxilary components necessary for operating the present invention.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 shows schematic representation of IgGI and scFv wherein Fab iscomposed of L-chain, and VH and CH1 domains, and scFv consists of VHdomain and VL domain which are linked by linker.

FIG. 2 shows illustration of pSEL preparation process.

FIG. 3 shows flow cytometric analysis of an expression of M21 chimeraantibody on COS7 cells. Control: COS7 cells without transformation ofplasmid. COS(pSELM21): COS7 cells with transformation of pSELM21.

FIG. 3, 1) shows staining with goat anti-human immunoglobulin antibody.Horizontal axis indicates amount of human immunoglobulin expressed onCOS7 cells. About 35% of COS7 cells express M21 chimera antibody.

FIG. 3, 2) shows antigen (GK1.5)-binding activity of the expressed M21chimera antibody. About 35% of COS7 cells show GK1.5-binding activity,from which the expressed M21 chimera antibody has binding activity forantigen GK1.5.

FIG. 4 shows an illustration for production of pSE.

FIG. 5 shows an illustration for production of pSE2.

FIG. 6 shows flow cytometric analysis of binding activity for yHBs withexpressed clone 2 and clone 3 in COS cells. Control: COS7 cells withouttransformation of the plasmid; COS (pSE clone no.2): COS cellstransformed with clone 2; and COS (pSE clone no. 3): COS cellstransformed with clone 3. The y-axis indicates number of cells and thex-axis indicates amount of human anti-HBs antibody expressed on COS7cells. 12.7% of COS7 cells in which clone 2 is transformed, expresshuman anti-yHBs antibody, and 6.6% of COS7 cells in which clone 3 istransformed, express human anti-yHBs antibody.

FIG. 7 shows results of competitive inhibition in use of clone 2 andclone 3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Following examples illustrate the present invention but are notconstrued as limiting.

EXAMPLE 1

(Preparation of pBR322 series membrane binding antibody expressionplasmid pSEL)

Human Cγ1 gene, to which a nucleotide sequence coding for human H-chainsignal sequence at 5′ terminus and nucleotide sequence coding fortransmembrane domain of human thrombomodulin at 3′ terminus wereligated, was inserted into a plasmid pSR, 3.1 kb, having ori from SV40,SRα promoter, polyadenylation signal and cloning sites of HindIII andXbaI, originated from pBR322 to construct a plasmid pSR-GM (4.7 kb).ApaI site located in the neighbouring with 5′ terminus of Cγ1 was usedfor cloning site in the 3′ terminus of H-chain variable regionnucleotide sequence. BamHI site was introduced in the 5′ terminus ofH-chain variable region nucleotide sequence. Introduction of BamHI sitewas performed by the following process. HindIII site and BamHI site wereintroduced in the 5′ terminus and 3′ terminus, respectively, ofnucleotide sequence coding for the region from H-chain signal peptide toN-terminal of H-chain variable region to synthesize the nucleotidefragment. The fragment was then temporarily inserted in the plasmid DNAin which Cγ1 and TM were previously inserted. A DNA fragment containingH-chain signal peptide, Cγ1 and TM, in this order, was obtained bycutting at HindIII site and XbaI site located in the 3′ side of TM. Thethus obtained DNA fragment was inserted in pSR which was cut at HindIIIsite and XbaI site (pSR-GM).

Similarly, a plasmid pSR-K (3.9 kb) was constructed by inserting anucleotide sequence coding for human κ chain signal sequence and humanCκ gene in pSR. Cloning site was constructed by newly inserting XhoIsite in the 5′ terminus and SpeI site at the neighbouring of 5′ terminusof Cκ in the 3′ terminus. For introduction of cloning site, HindIII siteand XhoI site were introduced in the 5′ terminus and 3′ terminus,respectively, of the nucleotide sequence coding for the region fromL-chain signal peptide to N-terminal of L-chain variable region tosynthesize the nucleotide fragment. The thus totally synthesizednucleotide fragment was temporarily inserted in the plasmid DNA. Cκ, forwhich SpeI site had been introduced by mutation, was previouslyincorporated in the said DNA. DNA fragment containing L-chain signalpeptide and Cκ was cut at the HindIII site and the XbaI site in the 3′side of Cκ, and was inserted in pSR which was cut at HindIII and XbaIsites (pSR-K). The pSR-K was digested by SalI (Takara Shuzo Co.hereinafter designates as Takara). After preparing blunt end by treatingwith a commercially available kit (DNA blunting kit, Takara), afragment, 2.0 kb, containing Cκ gene was isolated and extracted byagarose gel electrophoresis. The fragment was digested with ClaI(Takara) and ligated with pSR-GM to prepare pSEL. (FIG. 2)

EXAMPLE 2

(Preparation of pUC18 sesries membrane binding antibody expressionplasmid pSE)

A plasmid pEN-GM (5.3 kb) was prepared by inserting a HindIII-XbaIfragment containing human Cγ1 gene in pSR-GM, 1.6 kb into the HindIIIand XbaI cloning sites of a plasmid pEN, 3.7 kb, originated from pUC18,having ori from SV40, SRα promoter and polyadenylation signal. A plasmidpEN-K (4.5 kb) was prepared by inserting a HindIII-XbaI fragment (0.8kb) containing human Cκ gene of pSR-K in pEN.

The pEN-K was digested by NheI (Takara) and SmaI (Takara) to prepareblunt end, and a fragment (1.8 kb) containing Cκ gene was separated andextracted by agarose gel electrophoresis. The fragment was ligated withpEN-GM which was digested by NheI and blunt-ended. The plasmid pSC inwhich direction of transcription of Cκ gene was identical with that ofCγ1 gene, was selected from the thus prepared plasmid. For introducingnew EcoRI site in the downstream region between the two cloning sites ofH-chain variable region of pSC, a fragmnet within BamHI-ApaI in pSR-GMwas amplified by PCR in use of a primer 1 (5′-GTCCCAGGATCCCCGG-3′) (SEQID NO:1) and a primer 2(5′-CCGATGGGCCCTTGGTGGAGGCTGAATTCACGGTGACCGTGGTCC-3′) (SEQ ID NO:2). PCRwas performed by the conditions at 94° C. for 1 min.→at 55° C. for 2min.→at 72° C. for 2 min. in 25 cycles. The thus obtained amplifiedfragments were digested with BamHI (Takara) and ApaI (Takara) andinserted into pSC in place of the BamHI-ApaI sequence in pSC. Newlyprepared plasmid having the cloning site of BamHI and EcoRI in H-chainvariable region is designated as pSE. (FIG. 4)

EXAMPLE 3

(Preparation of pSE2 by modification of pSE)

A plasmid was prepared by changing BamHI and EcoRI of cloning sites inH-chain variable region of pSE to ClaI and MluI sites, and introducingSplI site in a boundary between L-chain variable region and Cκ gene as adownstream cloning side of L-chain variable region.

In order to replace the cloning site of H-chain variable regionnucleotide sequence, a HindIII-ApaI fragment, approximately 80 bp,containing H-chain signal sequence of pSE was amplified by PCR in use ofa primer HS (5′-TTTTAAGCTTGAACATGAAACACCTGTGGTT-3′) (SEQ ID NO:3) and aprimer HC(5′-CGATGGGCCCTTGGTGGGAGGCTGACGCGTTATAATCGATTGGGACAGGACCCTGACATCTGGGAGCTG-31′) (SEQ ID NO:4). Condition of PCR was at 94° C. for 1 min.→at 65°C. for 1 min.→at 72° C. for 1 min. in 25 cycles. The thus obtainedamplified fragments were digested by HindIII and ApaI. The digestedfragment was replaced by the sequence between HindIII-ApaI sites in pSEto prepare a plasmid pSE-CM having cloning sites of ClaI and MluI.

SplI site was newly introduced in use of synthetic oligonucleotide LS(5′-TCGAGAATTCGTACGGTGGCTGCACCA-3′) (SEQ ID NO:5) and LC(5′-CTAGTGGTGCAGCCACCGTACGAATTC-3′) (SEQ ID NO:6). A mixture ofequivalent amount of aqueous solution of LS and LC (1 mg/ml) in a tubewas immersed in water. After boiling the water, LS and LC were annealedby slightly cooling with standing at room temperature for about 3 hours.The thus generated LSLC mixture and pSE-CM treated with XhoI and SpeIdigestion were mixed at a molar ratio of 3:1 to ligate. Ligation KitVer. 2 (Takara) was used for the ligation. The new plasmid having SplIsite is designated as pSE2. (FIG. 5)

EXAMPLE 4

(Expression of M21 mouse monoclonal antibody on COS7 cells)

Mouse monoclonal antibody M21 (IgG1κ) is an antibody for rat IgG2b. AcDNA was synthesized from mRNA extracted from M21 hybridoma. H-chain andκ chain variable region fragments of M21 were cloned by PCR in use ofthe cDNA as a template. Extraction of mRNA was performed in use of QuickPrep mRNA purification Kit (Pharmacia Inc.). The cDNA synthesis wasperformed by cDNA Synthesis Kit (Boehringer Mannheim A.G.). These arecommercially available kits and the procedures were performed accordingto the attached protocol.

Primers used for PCR amplification of H-chain variable region are shownin Table 1 and primers used for that of κ chain variable region areshown in Table 2. PCR was performed in use of GeneAmp PCR Reagent Kitwith AmpliTaq DNA Polymerase (Takara) and was conducted at 94° C. for 1min.→at 55° C. for 2 min.→at 72° C. for 2 min. in 35 cycles, in 1.0-2.0mM Mg²⁺. Result of PCR indicated that V_(H) and V_(K) genes of M21 weresupposed to belong to subgroups I(B) and III, respectively. PCR productof H-chain variable region was digested by BamHI and ApaI and insertedinto pSEL to prepare pSELM21VH. PCR product of κ chain variable regionwas digested by XhoI (Takara) and SpeI (Takara) and inserted intopSELM21VH to construct membrane-bound M21 chimera antibody expressionvector pSELM21.

Plasmid DNA of pSELM21 was transferred to COS7 cells by means ofelectroporation. Expression of M21 chimera antibody on COS7 cells wasanalyzed by flow cytometry. Gene transfer by electroporation wasperformed in use of Gene Pulser (Bio-Rad Corp.) as follows. SubculturedCOS7 cells with DMEM containing 10% FCS (GIBCO BRL Inc.) wereinoculated, 3×10⁶ cells/plate, into a dish, diameter 150 mm, on the daybefore gene transfer. Cultured COS7 cells were washed twice with 15 mlof PBS (−)(Flow Laboratories Inc.) and harvested from dish by adding 5ml of EDTA trypsin solution (CosmoBio Inc.). Trypsin was inactivated byadding 3 ml of DME containing 10% FCS.

Cells were washed twice with 10 ml of previously cooled PS buffersolution (272 mM sucrose, 1 mM MgCl₂ and 7 mM sodium phosphate pH 7.4),and suspended at 8×10⁶ cells/ml in PS buffer solution. 0.5 ml of cellsuspension was set in Gene Pulser Cuvette (0.4 cm), and 5 μl of plasmidDNA (4 μg/ml distilled water) was added thereto and stirred. After seton ice for 5 minutes, the cells were twice pulsed at 3 μF. 500 V with 30sec. intervals. Further cells were set on the ice for 5 min.,transferred into dish, diameter 100 mm, and cultured in 10 ml of DMEMcontaining 10% FCS at 37° C. Temperature of the culture was set at 33°C. after 4 hours. Culture medium was exchanged on the next day to removedead cells and cultured for 60-72 hours. Cells were washed twice with 5ml of PBS (−). Previously ice-cooled 4 ml of 0.02% EDTA-PBS (−) wereadded in the dish, stood for 15 minutes at 4° C. and released cells wererecovered. Dish was washed with 1 ml 0.02% EDTA-PBS (−) and the washedsolution was combined with the recovered cells for analysis.

Expression of M21 mouse chimera antibody on the pSELM21 transformed COS7cells was determined. 50 μl of FITC-labelled goat anti-humanimmunoglobulin antibody (TAGO Inc: Cat. No. 2193), which was diluted50-fold with PBS(−) containing 1% BSA and 20% goat serum, were added tothe transformed cells and reacted for 30 minutes on the ice. Cells werespun down to remove supernatant, washed with 200 μl of 1% BSA-PBS(−) andfurther spun down to remove supernatant. This washing operation wasrepeated for 3 times. After washing, cells were suspended in 100 μl ofPBS (−) to prepare a sample for flow cytometry.

Binding activity of the expressed chimera antibody to antigen GK 1.5(rat IgG2b anti-mouse L3T4 antibody) was determined. 50 μl ofbiotin-labelled GK1.5, which was diluted 100-fold with 1% BSA-PBS (−),were added to the transformed cells 2×10⁵ and reacted for 30 min. on theice. Cells were spun down and washed three times. 50 μl of stocksolution of PE-labelled streptoavidin (Biomeda Ind: Cat. No. P22), whichwas diluted tenfold with 1% BSA-PBS (−), were added to the cells andreacted for 30 min. on the ice. Cells were spun down and washed threetimes. Washed cells were suspended in 100 μl of PBS (−) to prepare asample for flow cytometry.

Flow cytometric analysis was performed in use of EPICS Elite (CoulterInc.). Operation was performed according to the operational procedure.Result of flow cytometry is shown in FIG. 3. M21 chimera antibody wasexpressed on COS7 cells transformed with pSELM21. Also antigen (GK1.5)binding activity was maintained.

TABLE 1 Primer for amplifying mouse H-chain variable region nucleotidesequence Primer Sequence 1) Reverse primer (a primer at 5′ terminus:underline indicates BamHI site) M-1A GTCCCAGGAT  CCGCTTCAGG  AGTCAGGACCSEQ ID NO: 7               C      C    C C                             GM-1B GTCCCAGGAT  CCGCTGAAGG  AGTCAGGACC SEQ ID NO: 8              A     AC      T  C               T M-2AGTCCCAGGAT  CCGCTGCAAC  AATCTGGACC SEQ ID NO: 9              A  C TG    GC A  GC M-2BGTCCCAGGAT  CCACTGCAGC  AGCCTGGGGC SEQ ID NO: 10              C  A  AG   AT    AA               G  C              C                 T              T M-3AGTCCCAGGAT  CCGCTGGTGG  AATCTGGAGG SEQ ID NO: 11              C   A      G     G M-3B GTCCCAGGAT  CCGCTTCTCG  AGTCTGGAGGSEQ ID NO: 12                T  A  C      A M-3CGTCCCAGGAT  CCGCTTGAGG  AGTCTGGAGG SEQ ID NO: 13                 G TT     A   AG M-3D GTCCCAGGAT  CCGCTGGTGG  AGTCTGGGGGSEQ ID NO: 14               CG A            A                T T M-6GTCCCAGGAT  CCGCTCGTGG  AGTCTGGGGG SEQ ID NO: 15              C  GT       A   CA M-7 GTCCCAGGAT  CCGTTGGTAC  AGTCTGGACCSEQ ID NO: 16                     G          T 2) Forward primer (aprimer at 3′ terminus: underline indicates ApaI site) MG1-1ACAGATGGGC  CCGTCGTTTT  GGCTGAGGAG  A SEQ ID NO: 17                           C                            G MG1-2ACAGATGGGC  CCGTCGTTTT  GGCTGCAGAG  A SEQ ID NO: 18                           A                            C

TABLE 2 Primer for amplifying mouse κ chain variable region PrimerSequence 1) Reverse primer (a primer at 5′ terminus: underline indicatesXhoI site SEQ ID NOS: 19-30) MK5-1 TGTGCCCTCG  AGATGACACA  GTCTCCATCC  TSEQ ID NO: 19                  T T       G   A A                                 T MK5-2ATGTGCCCTCG  AGATGACCCA  AACTCCACTC  TC SEQ ID NO: 20              G    T    G T         A MK5-2BTGTGCCCTCG  AGATGACGCA  GGCTGCATTC  T SEQ ID NO: 21                   T          CCC MK5-2CTGTGCCCTCG  AGATAACCCA  GGATGAACTC  TC SEQ ID NO: 22                G G      A                   T MK5-3TGTGCCCTCG  AGCTGACCCA  ATCTCCAGCT                    A    G     T MK5-4TGTGCCCTCG  AGCTCACCCA  GTCTCCAGC                                AMK5-5A TGTGCCCTCG  AGATGACACA  GACTACATCC  TC                               A MK5-5BTGTGCCCTCG  AGATGACACA  GTCTCCATCC  T                 A  C             TMK5-5C TGTGCCCTCG  AGATGACTCA  GTCTCCAGCC              C A  C          T MK5-5DTGTGCCCTCG  AGATGACCCA  GTCTCCCAAA  TCC                        AA   AA      T MK5-5ETGTGCCCTCG  AGGTGACCCA  GTCTCCAGCA MK5-6TGTGCCCTCG  AGCTCACCCA  GTCTCCAGC                  T T 2) Forward primer(a primer at 3′ terminus: underline indicates SpeI site)(SEQ ID NO: 31)MK3-2 ATGGATACTA  GTGGTGCAGC  ATCAGCCC

EXAMPLE 5

(Preparation of mouse antibody variable region plasmid library)

Spleen of BALB/c mouse, female, was dissected and hemolysis was made byadding 5 ml of hemolytic buffer (NH₄ Cl 8.29 g/l, KHCO₃ 1.0 g/l, EDTA3.67 g/l, pH 7.4), washed twice with 10 ml of PBS(−) to preparelymphocytes. The mRNA was extracted from the lymphocytes and cDNA wassynthesized to prepare a template for PCR. Extraction of mRNA wasperformed in use of mRNA purification kit (Pharmacia Inc.) and synthesisof cDNA was made in use of cDNA synthesis kit (Boehringer MannheimA.G.). PCR was perfomed in use of the primers shown in Tables 1 and 2 toprepare H-chain and κ chain variable region fragments in each subgroup.In mouse, κ chain is a major of L-chain, consequently only κ chain wasprepared as a L-chain. Since a complementary sequence from 3′ terminusof J_(H) gene to 5′ terminus of Cγ₁ in mouse was used as a forwardprimer, preparation of H-chain was limited in a variable region of IgG1subclass antibody. In each of H-chain abed κ chain, each subgroup wasmixed in equivalent to use in the forthcoming operation. XhoI site andSpeI site were introduced in the reverse primer and the forward primer,respectively, in the κ chain variable region. A PCR product of the κchain variable region was inserted into pSEL in these restriction enzymesites to prepare mouse κ chain variable region library (MVKL).Similarly, the H-chain variable region fragment was inserted into MVKLat BamHI site in the reverse primer and ApaI site in the forward primerto prepare mouse antibody variable region library (MVL).

EXAMPLE 6

(A trial experiment for concentrating antibody variable region fragmentwith specific antigen binding-activity)

M21 mouse anti-rat IgG2b antibody (IgG1κ) was used as a model antibody.A trace amount of pSELM21 prepared in Example 4 was mixed to MVLprepared in Example 5 and the mixture was introduced into COS7 cells byelectroporation. The possibilities on selective concentration of theCOS7 cells per se which expressed M21 chimera antibody from COS7 cellsexpressing various antibodies were examined. COS7 cells transformed withthe plasmid were stained by biotin-labelled GK1.5 (rat IgG2b) andPE-labelled streptoavidin and sorting out the cells in use of EPICSElite. Positive cells were sorted from approximately 6.4×10⁶ cells.Plasmid DNA was recovered from the separated COS7 cells by Hirt methodand introduced into E. coli DH5. Competent High E. coli DHS (TOYOBO) wasused in the transformation according to the attached protocol. Appearedcolonies were transferred to nylon membrane (Hybond-N, Amersham Inc.),and subjected to colony hybridization in use of M21 H-chain variableregion nucleotide sequence specific oligonucleotide probe (M21H2:5′-GTAGGAGAGGCTTATTACTA-3′) (SEQ ID NO: 32) and κ chain variable regionnucleotide sequence specific oligonucleotide probe (M21K2:5′-AAGTATGCATCCAACCTAGA-3′) (SEQ ID NO: 33) to count a ratio of pSELM21.Colony hybridization was performed in use of the probes with labelleddigoxigenin (DIG oligo-nucleotide 3′ endolabelling kit was used.Boehringer Mannheim A.G.: Cat. No. 1362372). Detection of probe wasperformed in use of DIG Luminescent Detection kit for nucleic aciddetection (Boehringer Mannheim Cat. No. 1363514). The operation wasfollowed according to the protocol. Double positive colonies in thesetwo probes were determined to be the transformant containing pSELM21.Rate of concentration was calculated by a change in the ratio of pSELM21transformant in the total transformants in pre-and post-sorting. Inhybridization, double positive colonies in the frequency of 2/2107 couldbe concentrated to 54-fold in that of 10/195. (Table 3)

TABLE 3 Total No. No. of double Concentration Sorting of colony positivecolonies rate before 2107  2 after  195 10 54

EXAMPLE 7

(Preparation of human antibody variable region plasmid library)

Hepatitis B vaccine (Bimmugen)(Chemotherapy and Serotherapy LaboratoriesInc.) was administered in a volunteer with positive anti-HBs antibodyand peripheral blood 150 ml was collected after 6 days. After lymphocytefraction was prepared by conventional method using Ficoll, mRNA wasextracted and cDNA was synthesized to prepare template for PCR.Lymphocytes preparation using Ficoll is described in the reference (NewBiochemistry Experiments Series 12: Molecular Immunology, Chapter 1,Isolation of lymphocytes, by K. Nishikawa). Extraction of mRNA wasperformed in use of mRNA purification kit (Pharmacia Inc.) and synthesisof cDNA was made in use of cDNA synthesis kit (Pharmacia Inc.). Since,in the present experiment, gene source was selected from the subjectwith high anti-HBs antibody titer, and anti HBs antibody wouldsufficiently be contained in a library containing merely κ chain inL-chain, only κ chain was selected in L-chain in the present library.PCR was performed in use of specific primer for each subgroup in Tables4 and 5. Conditions on PCR for H-chain variable region nucleotidesequence: at 94° C. for 1 min.→at 69° C. for 1 min.→at 72° C. for 1min., in 30 cycles, Mg²⁺ conc. 0.5 mM. Referring to number of germ lineV_(H) gene contained in each subgroup, the amplified products by PCRfrom each subgroup of H-chain variable region nucleotide sequence weremixed together at the ratio of I:II:III:=2:3:4. In κ chain variableregion nucleotide sequence, PCR products of each subgroup were mixedtogether at a ratio of I:IIA:IIB:III:IV=15:8:2:5:1.

XhoI site and SpeI site were introduced in the reverse primer and theforward primer for amplifying κ chain variable region nucleotidesequence, respectively. Then PCR product of κ chain variable region wasdigested by these restriction enzymes and ligated into pSE digested bythe same enzyme. Ligated product was recovered from ethanolprecipitation and was introduced into E. coli DH5 by electroporation.Electroporation was conducted by E. coli Pulser, Bio-Rad Inc. accordingto the attached protocol. Transformed E. coli was spreadon a LB platecontaining ampicillin and cultured for overnight. The colonies(approximately 7×10⁴ cells) were recovered and cultured in terrificbroth for overnight. Plasmid DNA was extracted and purified to preparehuman Vκ gene library (pSEhVKmix).

BamHI site and EcoRI site in the reverse primer and the forward primerfor amplifying H-chain variable region nucleotide sequence,respectively, were used for insertion in pSE. Colonies, approx. 5×10⁵,obtained from introduction of the ligated product into DH5 wererecovered and cultured. Plasmid DNA was extracted to prepare human VHgene library (pSEhVHmix). The pSEhVKmix was digested by XhoI and SpeI torecover κ chain variable region fragment. This was ligated with pSEhVHdigested by XhoI and SpeI. The ligated product was introduced into DH5,and the thus obtained approx. 5×10⁶ colonies were recovered. Plasmid DNAwas extracted from the cultured cells and purified to prepare humanantibody variable region plasmid library (PSEhVmix).

TABLE 4 Primer for amplifying human H-chain variable region nucleotidesequence Primer Sequence 1) Reverse primer (5′ terminal primer:underline indicates BamHI site SEQ ID NOS: 34-36 SEH5-1GGGGGGATCC  GCTGGTGCAG  TCCGGACCAG  AGGTG SEQ ID NO: 34               T           T  GG T SEH5-2GGGGGGATCC  GCTACAGCAG  TCAGGCCCAG  GACTG SEQ ID NO: 35               G   G     GG  TG SEH5-3GGGGGGATCC  GCTGGTGGAG  TCTGGAGGAG  ACGT SEQ ID NO: 36                T            G      G T 2) Forward primer (3′ terminalprimer: under1ine indicates EcoRI site (SEQ ID NO: 37) SEH3GGGGGAATTC  ACAGTGACCA  GGGTCCCACG  CCC              G      G  TT  G  CT   G                             T  T

TABLE 5 Primer for amplifying human κ chain variable region PrimerSequence Reverse primer (5′ terminal primer: underline indicates XhoIsite)(SEQ ID NOS: 38-42) SEK5-1 GGGGCTCGAG  ATGACCGAGT  CTCCATCCAC  ACTGSEQ ID NO: 38             G               T  TT   C SEK5-2AGGGGCTCGAG  ATGACCCAGA  CTCCACTCTC  CCTG SEQ ID NO: 39                 T   T              T SEK5-2BGGGGCTCGAG  ATGACCCAGA  CTCCACTCTC  CTCA SEQ ID NO: 40 SEK5-3GGGGCTCGAG  ATGACGCAGT  CTCCAGCCAC  CC SEQ ID NO: 41            T                 G SEK5-4GGGGCTCGAG  ATGACCCAGT  CTCCAGACTC  CCTG SEQ ID NO: 42 2) Forward primer(3′ terminal primer: underline indicates SpeI Site)(SEQ ID NO: 43) SEK3GGGGACTAGT  GGTGCAGCCA  CAGTACGTTT  AAT                                    G

EXAMPLE 8

(Screening for human anti-HBs antibody variable region nucleotidesequence)

The fact that anti-HBs antibody variable region nucleotide sequence canbe screened from pSEhVmix prepared in Example 7, has been confirmed.Antigen, yHBs antigen for research studies, was purchased fromChemotherapy and Serotherapy Institute Inc. A yHBs antigen wasbiotinylated in K.K. Immunology and Biology Institute and used as anantigen for screening. The pSEhVmix was introduced into COS7 cells bymeans of electroporation to prepare membrane-bound human antibodyexpressing COS7 cells library. DNA 20 μg of pSEhVmix was mixed with4×10⁶ COS7 cells (500 μl), and pulsed twice at 3 μF, 450 V. Thisoperation was repeated 3 times to prepare COS7 cells library. Afterculturing the cells in DMEM containing 10% FCS for 60 hours, COS7 cellswere harvested from culture dish by adding 0.02% EDTA-PBS (−).

Biotin-labelled yHBs was diluted to 1 μg/ml with 1% BSA-PBS to use forstaining of COS7 cells library. In the secondary staining PE-labelledstreptoavidin (Biomeda Inc: Cat. No. P22) was used. Details of stainingmethod were described in Example 4. Sorting of the stained COS7 cellslibrary was performed by use of FACS Vantage attached with Macro SORTsystem, Becton Deckinson Inc. Sorting operation was conducted accordingto the attached protocol to recover PE-positive cell fraction. PlasmidDNA was recovered by Hirt method from the thus obtained PE-positive cellfraction. A half thereof was introduced into E. coli DH5 by means ofelectroporation. Colonies appeared on the LB plate containing 0.1%ampicillin were picked up and cultured in terrific broth for overnight,and the plasmid DNA was extracted from the culture. These concentrationoperations were repeated for 3 times. The plasmid DNA was introducedinto E. coli DH5 by electroporation. Plasmid DNA of 38 clones in theappeared colonies was purified, and H-chain and κ chain variable regionnucleotide sequence were analyzed. Sequences were analyzed in use of DNAsequencer ver. 1. 2. 0. Model 373A (Applied Biosystems Inc.) accordingto the attached protocol. Labelling reaction was performed with primersSEQHC (5′-CTCTTGGAGGAGGGTGCCAG-3′) (SEQ ID NO:44) for H-chain and SEQLC(5′-CCAGATTTCAACTGCTCATCAGA-3′) (SEQ ID NO:45) for κ chain in use ofPRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit (AppliedBiosystems Inc.). The operation was conducted according to the attachedprotocol.

Affinity of yHBs antigen for antibodies which were expressed on COS7cells transformed with isolated plasmid DNA was examined. Resultindicated that 29 clones in 38 clones tested bound to yHBs antigen, andthe bound clones were classified in 5 indepndent clones. Result ofstaining of 2 clones in the 5 clones is shown in FIG. 6. Result ofcompetitive inhibition in use of competitor yHBs without biotinylationis shown in FIG. 7. COS7 cells transformed with plasmid were reactedwith various concentrations of the competitor on the ice for 30 minutes,and biotin-labelled yHBs was added at 1 μg/ml thereto, then the cellswere stained conventionally. In FIG. 7, per centinhibition is shown bysetting a ratio (%) of cell fraction with positive staining as 100% atthe competitor concentration 0. As shown in FIG. 7, these 2 clonesrecognize yHBs specifically.

In the present invention, a possibility of hitherto unknown screeningmethod for antibody variable region nucleotide sequence in use ofeukaryotic cell expression system is provided. According to the presentinvention, variable region nucleotide sequence of antigen-specificantibody can effectively be selected with maintaining exact propertiesof antibody produced by animal cells. Human monoclonal antibody for anyantigens can also be produced.

45 16 base pairs nucleic acid single linear cDNA unknown 1 GTCCCAGGATCCCCGG 16 45 base pairs nucleic acid single linear cDNA unknown 2CCGATGGGCC CTTGGTGGAG GCTGAATTCA CGGTGACCGT GGTCC 45 31 base pairsnucleic acid single linear cDNA unknown 3 TTTTAAGCTT GAACATGAAACACCTGTGGT T 31 69 base pairs nucleic acid single linear cDNA unknown 4CGATGGGCCC TTGGTGGGAG GCTGACGCGT TATAATCGAT TGGGACAGGA CCCTGACATC 60TGGGAGCTG 69 27 base pairs nucleic acid single linear cDNA unknown 5TCGAGAATTC GTACGGTGGC TGCACCA 27 27 base pairs nucleic acid singlelinear cDNA unknown 6 CTAGTGGTGC AGCCACCGTA CGAATTC 27 30 base pairsnucleic acid single linear cDNA unknown 7 GTCCCAGGAT CCGCTTCAGGAGTCAGGACC 30 30 base pairs nucleic acid single linear cDNA unknown 8GTCCCAGGAT CCGCTGAAGG AGTCAGGACC 30 30 base pairs nucleic acid singlelinear cDNA unknown 9 GTCCCAGGAT CCGCTGCAAC AATCTGGACC 30 30 base pairsnucleic acid single linear cDNA unknown 10 GTCCCAGGAT CCACTGCAGCAGCCTGGGGC 30 30 base pairs nucleic acid single linear cDNA unknown 11GTCCCAGGAT CCGCTGGTGG AATCTGGAGG 30 30 base pairs nucleic acid singlelinear cDNA unknown 12 GTCCCAGGAT CCGCTTCTCG AGTCTGGAGG 30 30 base pairsnucleic acid single linear cDNA unknown 13 GTCCCAGGAT CCGCTTGAGGAGTCTGGAGG 30 30 base pairs nucleic acid single linear cDNA unknown 14GTCCCAGGAT CCGCTGGTGG AGTCTGGGGG 30 30 base pairs nucleic acid singlelinear cDNA unknown 15 GTCCCAGGAT CCGCTCGTGG AGTCTGGGGG 30 30 base pairsnucleic acid single linear cDNA unknown 16 GTCCCAGGAT CCGTTGGTACAGTCTGGACC 30 31 base pairs nucleic acid single linear cDNA unknown 17ACAGATGGGC CCGTCGTTTT GGCTGAGGAG A 31 31 base pairs nucleic acid singlelinear cDNA unknown 18 ACAGATGGGC CCGTCGTTTT GGCTGCAGAG A 31 31 basepairs nucleic acid single linear cDNA unknown 19 TGTGCCCTCG AGATGACACAGTCTCCATCC T 31 32 base pairs nucleic acid single linear cDNA unknown 20TGTGCCCTCG AGATGACCCA AACTCCACTC TC 32 31 base pairs nucleic acid singlelinear cDNA unknown 21 TGTGCCCTCG AGATGACGCA GGCTGCATTC T 31 32 basepairs nucleic acid single linear cDNA unknown 22 TGTGCCCTCG AGATAACCCAGGATGAACTC TC 32 30 base pairs nucleic acid single linear cDNA unknown23 TGTGCCCTCG AGCTGACCCA ATCTCCAGCT 30 29 base pairs nucleic acid singlelinear cDNA unknown 24 TGTGCCCTCG AGCTCACCCA GTCTCCAGC 29 32 base pairsnucleic acid single linear cDNA unknown 25 TGTGCCCTCG AGATGACACAGACTACATCC TC 32 31 base pairs nucleic acid single linear cDNA unknown26 TGTGCCCTCG AGATGACACA GTCTCCATCC T 31 30 base pairs nucleic acidsingle linear cDNA unknown 27 TGTGCCCTCG AGATGACTCA GTCTCCAGCC 30 33base pairs nucleic acid single linear cDNA unknown 28 TGTGCCCTCGAGATGACCCA GTCTCCCAAA TCC 33 30 base pairs nucleic acid single linearcDNA unknown 29 TGTGCCCTCG AGGTGACCCA GTCTCCAGCA 30 29 base pairsnucleic acid single linear cDNA unknown 30 TGTGCCCTCG AGCTCACCCAGTCTCCAGC 29 28 base pairs nucleic acid single linear cDNA unknown 31ATGGATACTA GTGGTGCAGC ATCAGCCC 28 20 base pairs nucleic acid singlelinear cDNA unknown 32 GTAGGAGAGG CTTATTACTA 20 20 base pairs nucleicacid single linear cDNA unknown 33 AAGTATGCAT CCAACCTAGA 20 35 basepairs nucleic acid single linear cDNA unknown 34 GGGGGGATCC GCTGGTGCAGTCCGGACCAG AGGTG 35 35 base pairs nucleic acid single linear cDNAunknown 35 GGGGGGATCC GCTACAGCAG TCAGGCCCAG GACTG 35 34 base pairsnucleic acid single linear cDNA unknown 36 GGGGGGATCC GCTGGTGGAGTCTGGAGGAG ACGT 34 33 base pairs nucleic acid single linear cDNA unknown37 GGGGGAATTC ACAGTGACCA GGGTCCCACG CCC 33 34 base pairs nucleic acidsingle linear cDNA unknown 38 GGGGCTCGAG ATGACCCAGT CTCCATCCAC ACTG 3434 base pairs nucleic acid single linear cDNA unknown 39 GGGGCTCGAGATGACCCAGA CTCCACTCTC CCTG 34 34 base pairs nucleic acid single linearcDNA unknown 40 GGGGCTCGAG ATGACCCAGA CTCCACTCTC CTCA 34 32 base pairsnucleic acid single linear cDNA unknown 41 GGGGCTCGAG ATGACGCAGTCTCCAGCCAC CC 32 34 base pairs nucleic acid single linear cDNA unknown42 GGGGCTCGAG ATGACCCAGT CTCCAGACTC CCTG 34 33 base pairs nucleic acidsingle linear cDNA unknown 43 GGGGACTAGT GGTGCAGCCA CAGTACGTTT AAT 33 20base pairs nucleic acid single linear cDNA unknown 44 CTCTTGGAGGAGGGTGCCAG 20 23 base pairs nucleic acid single linear cDNA unknown 45CCAGATTTCA ACTGCTCATC AGA 23

What is claimed is:
 1. An expression vector having restriction sites toreceive DNA encoding the variable regions of antibodies, said vectorexpressing polypeptides comprising said variable regions in themembrane-bound form on the surface of eukaryotic cells, and said vectorbeing replicable in said cells, said vector containing at least one ofthe nucleotide sequences AKL and AKH:5′-P_(L)-S_(L)-C_(L)-M_(L)-A_(L)-3′  (AKL)5′-P_(H)-S_(H)-C_(H)-M_(H)-A_(H)-3′  (AKH) wherein P_(L) and P_(H) eachrepresent a promoter; S_(L) and S_(H) each represent a nucleotidesequence coding for a signal peptide; C_(L) represents a nucleotidesequence coding for the L-chain constant region of an antibody; C_(H)represents a nucleotide sequence coding for the H-chain constant regionof an antibody or a nucleotide sequence coding for the C_(H1) portion ofthe H-chain constant region of an antibody; A_(L) and A_(H) eachrepresent a polyadenylation signal; and at least one of M_(L) and M_(H)represents a nucleotide sequence coding for a transmembrane domain,wherein one is M_(L) and M_(H) is a phosphodiester bond when theexpression vector contains both AKL and AKH, and wherein AKL and AKH arefree of nucleotide sequences encoding antibody variable regions andcontain said restriction sites comprising sites R1_(L), R2_(L), R1_(H)and R2_(H) associated with S_(L), C_(L), S_(H) and C_(H) respectively,to facilitate insertion of nucleotide sequences coding for the L-chainand H-chain variable regions between S_(L) and C_(L), and S_(H) andC_(H), respectively.
 2. The expression vector according to claim 1wherein said cloning sites R1_(L), R2_(L), R1_(H) and R2_(H) areselected from recognition sequence of restriction enzymes MunI, AclI,BspLU11I, MluI, BssHII, NheI, XbaI, SplI, BspI407I, ClaI, XhoI, SalI andAf1II.
 3. The expression vector according to claim 1 wherein saidtransmembrane domain is the transmembrane domain of thrombomodulin. 4.The expression vector according to claim 3 wherein said expressionvector contains AKL and AKH, and the cloning sites R1_(L), R2_(L),R1_(H) and R2_(H) are the recognition sequences of XhoI, SplI, ClaI andMluI, respectively, and M_(L) is a phosphodiester bond.
 5. Theexpression vector according to claim 3 wherein said expression vectorcontains AKL and AKH, and the cloning sites R1_(L), R2_(L), R1_(H) andR2_(H) are the recognition sequences of XhoI, SpeI, BamHI and EcoRI,respectively, and M_(L) is a phosphodiester bond.
 6. The expressionvector according to claim 3 wherein said expression vector contains AKLand AKH, and the cloning sites R1_(L), R2_(L), R1_(H) and R2_(H) are therecognition sequences of XhoI, SpeI, BamHI and ApaI, respectively, andM_(L) is a phosphodiester bond.
 7. The expression vector according toclaim 1 wherein the said vector is replicable in COS cells.
 8. Theexpression vector of claim 1 further comprising a nucleotide sequencecoding for H-chain variable region of antibodies or a nucleotidesequence coding for L-chain variable region of antibodies inserted intothe cloning sites of said vector.
 9. A group of eukaryotic cellsexpressing polypeptides containing the H-chain and L-chain variableregions of antibodies in the membrane-bound form on the surface of thecells transfected with the vector of claim
 8. 10. A method for selectingnucleotide sequences coding for antibody variable regions binding to aspecific antigen from the nucleotide sequences coding for a large numberof antibody variable regions, comprising: contacting the cells of claim9 with an antigen, isolating the cells bound to said antigen from theremaining cells, and recovering the expression vector from the isolatedcells to obtain nucleotide sequences coding for antibody variableregions bound to the said antigen.
 11. The method according to claim 10comprising immobilizing the antigen on a solid surface.
 12. The methodaccording to claim 10 comprising labelling the antigen with fluorescentsubstance, biotin or magnetic beads.
 13. A plasmid vector pSEL (FERMBP-4896).
 14. A plasmid vector pSE (FERM BP-4894).
 15. A plasmid vectorpSE2 (FERM BP-4895).
 16. The method according to claim 10 wherein thespecific antigen is hepatitis B antigen.
 17. A kit for screeningnucleotide sequences coding for variable regions of the antigen-specificantibody comprising the expression vector of claim 1, host cells andauxiliary components.